Cross-talk-free Fluorescence Cross-Correlation Spectroscopy by the Switching Method

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Fluorescence cross-correlation spectroscopy (FCCS) is used as a powerful technique to analyze molecular interactions both <i>in vitro</i> and <i>in vivo</i>. This method basically requires two laser excitations for two target molecules labeled with fluorophores of different colors. Their coincidence in a microscopic detection volume is analyzed using two detectors. Any overlap of emission spectra of the two fluorophores, however, gives rise to false-positive data about their interaction. To overcome this problem, we have developed a new FCCS system, in which two excitation lasers are switched alternately by modulation using an acousto-optic tunable filter (AOTF). In this report, we demonstrate the feasibility of switching FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP) with a cleavage site of caspase-3 inserted was expressed in HeLa cells, and proteolysis assay was performed during apoptotic cell death. Due to the absence of cross-talk signals, the FCCS measurement with the switching function gave a large change in relative cross-correlation amplitude after protein cleavage. Hence, switching FCCS enables more reliable measurement of molecular interactions than conventional FCCS.<br>


  • Cell Structure and Function

    Cell Structure and Function 33(1), 143-150, 2008

    Japan Society for Cell Biology


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